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A multidomain hub anchorsthe chromosome segregationand chemotactic machineryto the bacterial pole
Yoshiharu Yamaichi,
1,2,5
Raphael Bruckner,
1,2,5,6
Simon Ringgaard,
1,2
Andrea Mo ¨ ll,
1,2
D. Ewen Cameron,
2,7
Ariane Briegel,
3
Grant J. Jensen,
3,4
Brigid M. Davis,
1,2
and Matthew K. Waldor
1,2,4,8
1
Division of Infectious Diseases, Brigham and Women’s Hospital,
2
Department of Microbiology and Immunobiology, HarvardMedical School, Boston, Massachusetts 02115, USA;
3
Division of Biology, California Institute of Technology, Pasadena,California 91125, USA;
4
Howard Hughes Medical Institute, Boston, Massachusetts 02115, USA
The cell poles constitute key subcellular domains that are often critical for motility, chemotaxis, and chromosomesegregation in rod-shaped bacteria. However, in nearly all rods, the processes that underlie the formation,recognition, and perpetuation of the polar domains are largely unknown. Here, in
Vibrio cholerae
, we identifiedHubP (hub of the pole), a polar transmembrane protein conserved in all vibrios, that anchors three ParA-likeATPases to the cell poles and, through them, controls polar localization of the chromosome srcin, thechemotactic machinery, and the flagellum. In the absence of HubP,
oriCI
is not targeted to the cell poles,chemotaxis is impaired, and a small but increased fraction of cells produces multiple, rather than single, flagella.Distinct cytoplasmic domains within HubP are required for polar targeting of the three ATPases, whilea periplasmic portion of HubP is required for its localization. HubP partially relocalizes from the poles to themid-cell prior to cell division, thereby enabling perpetuation of the polar domain in future daughter cells. Thus,a single polar hub is instrumental for establishing polar identity and organization.
[
Keywords
:
Vibrio cholerae
; cell polarity; chemotaxis; chromosome segregation; motility]Supplemental material is available for this article.
Received June 29, 2012; revised version accepted August 27, 2012.
The formation and recognition of subcellular domainsis critical for numerous cellular processes, even in relativelysimple unicellular organisms such as bacteria (Shapiroet al. 2009; Rudner and Losick 2010; Thanbichler 2011).Without an ability to establish intracellular landmarks,key events in the bacterialcell cycle, such aschromosomesegregation and formation of the cell division plane, areinstead subject to stochastic variation that can lead todetrimental results. Bacterial morphology and motilityalsooftendependonpositionalrecognition;e.g.,forcorrectlocalizationofpiliandflagella.Afewdeterminantsofsuchsubcellular patterns have been well studied, such asMinCDE, which enable a number of bacterial species toform a division plane at the mid-cell (Raskin and de Boer1997; Lutkenhaus 2007). In Gram-positive bacteria andactinomycetes, DivIVA recruits other proteins to the cellpoles and septum and thereby regulates several cell pro-cesses (Marston et al. 1998; Ben-Yehuda et al. 2003; Fla¨rdh2010).Furthermore,inthedifferentiating
a
-proteobacterium
Caulobacter crescentus,
TipN and PopZ are markers anddeterminantsofpoleformation(Huitemaetal.2006;Lamet al. 2006; Bowman et al. 2008; Ebersbach et al. 2008).However,inmostbacteria,themeansbywhichsubcellulardomains are generated or recognized remain unknown.In the Gram-negative rod
Vibrio cholerae
, as in otherrod-shaped bacteria, the cell poles are key subcellulardomains, and the bacterium has the ability to distinguishpoles from sides and the old pole from the new (mostrecently generated) pole. For example, following cell di-vision, the srcin region of the larger of
V. cholerae
’s twochromosomes (
oriC
of chrI,
oriCI
) is always found at theold pole, as is an array of chemoreceptors with associated
5
These authors contributed equally to this work.Present addresses:
6
Department of Cell Biology, Harvard Medical School,240 Longwood Avenue, Boston, MA 02115, USA;
7
Department of Bio-medical Engineering, 44 Cummington Street, Boston University, Boston,MA 02215, USA.
8
Corresponding authorE-mail mwaldor@rics.bwh.harvard.edu
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.199869.112.
2348 GENES
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DEVELOPMENT 26:2348–2360
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2012 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/12; www.genesdev.org
chemosignaling proteins, and the bacterial flagellum alsodevelops at this site. We found that targeting of
oriCI
tothe pole is dependent on ParA1 (Fogel and Waldor 2006),a homolog of bacterial plasmid partitioning proteins, andthat the localization of the chemotactic apparati is alsoregulated by a ParA homolog, ParC (Ringgaard et al. 2011).FlagellarassemblyisregulatedbyathirdParA-likeprotein,FlhG(Correaetal. 2005; Kusumotoet al.2008),raisingthepossibility that these proteins rely on related processes togovern spatial patterning. Notably, each protein formsa focus at the old pole in newborn cells, although theirdistribution is not always limited to this site (Fogel andWaldor 2006; Kusumoto et al. 2008; Ringgaardet al. 2011).As the cell cycle progresses and the cell prepares fordivision, which will yield a ‘‘new’’ old pole in one of thedaughter cells, ParA1, ParC, and FlhG all adopt a bipolarpattern.However,themeansbywhichtheirdistributionisinitiallydeterminedandsubsequentlyshiftedhasnotbeenestablished.
V. cholerae
(a
g
-proteobacterium) lacks appar-ent homologs of TipN and PopZ; in fact, these polardeterminants are restricted to a subset of
a
-proteobacteria(Lam et al. 2006; Bowman et al. 2008; Ebersbach et al.2008).ParA family proteins are typically relatively small,cytoplasmic proteins that contain a deviant WalkerAAA ATPase motif. They often have the ability to self-associate; e.g., into polymers that may push or pull DNAand thereby promote its equal distribution betweendaughter cells. ParA family proteins have also been foundtogovernthelocalizationofthedivisionsite(e.g.,viaMinDand MipZ) (Thanbichler and Shapiro 2006; Lutkenhaus2007), type IV pili (e.g., via TadZ/CpaE) (Viollier et al.2002; Perez-Cheeks et al. 2012; Xu et al. 2012), andconjugative transfer machinery (via VirC1) (Atmakuriet al. 2007). They often have a dynamic distributionwithin the cell, and this distribution is key for properfunction. In most cases, the proteins’ ability to bind and/or hydrolyze ATP is critical for their subcellular localiza-tion and function (Szardenings et al. 2011; Lutkenhaus2012). It should be noted, however, that not all ParA-likeproteins, even in
V. cholerae,
form foci at the cell poles.The small chromosome (chrII)-associated protein ParA2and MinD are oscillatory and more diffusely distributedthroughout much of the cell (Raskin and de Boer 1999;Fogel and Waldor 2006). ParA proteins associated withplasmid segregation have also been observed to oscillate.The distinct localization patterns of these proteins arelikely determined by the specific protein partners withwhich each interacts.We performed a genetic screen to identify factors thatcontribute to the establishment of polar identity in
V. cholerae
and found that a single protein, VC0998(henceforth called HubP, for hub of the pole), is requiredfor theproperpolarlocalizationofParA1,ParC,andFlhG.In the absence of HubP,
oriCI
is not targeted to the cellpoles, chemotaxis is impaired due to the absence or mis-localization of polar chemotactic receptor arrays and theirassociated signaling proteins, and cells have an increasedpropensity to produce multiple, rather than single, flagella.HubP, like the three ParA family ATPases, is localized tothe cell poles; however, it routinely marks both poles—rather than the single (old) pole typically observed for theother proteins in young cells—and can migrate betweenthe two poles. Despite the homology between ParA1,ParC, and FlhG, their polar targeting by HubP appears torely on different mechanisms, and a distinct region of HubP is needed to localize each client protein. Thus,HubP is a multifaceted scaffold necessary for the estab-lishmentofpolaridentityandorganization;itisajunctionpointaroundwhichmultiplepolarprocessesareoriented.
Results
Identification of HubP, an organizer of polar featuresand processes
Our screen for pole-organizing factors, which was similarto a screen in
C. crescentus
(Huitema et al. 2006), waspremised on the idea that
V. cholerae
lacking a polarlandmark protein is likely to have impaired motility/chemotaxis, since its flagella and/or chemotaxis proteinsmay be mislocalized. Therefore, we focused our analysison genes that had previously been linked to a motilitydefect (Cameron et al. 2008) yet did not have an obviousconnection to chemotaxis or flagellar assembly. A set of 91 mutants was transformed with an expression constructforapreviouslydescribedpolarmarkerprotein,ParA1[K11E]-YFP, which, in contrast to wild-type ParA1-YFP, formsdiscrete bipolar foci throughout the cell cycle (Ringgaardet al. 2011). Transformants were individually screenedusingfluorescence microscopytoidentifystrainsinwhichthis protein is mislocalized.We found that disruption of a previously undescribedgene,
vc0998
(
hubP
), caused ParA1[K11E] to be diffuselydistributed throughout cells, rather than displaying itstypical bipolar distribution (Fig. 1A,E). This pattern wasobservedinbothatransposoninsertionmutant(
hubP
T
Tn
)and a strain with an in-frame deletion of
hubP
. Further-more, wild-type localization of ParA1[K11E] was re-stored in the
D
hubP
strain by expression of the proteinin
trans
, providing strong evidence that polar targetingof ParA1[K11E] is dependent on the presence of HubP.HubPis alsorequired for thepropercellular positioningof several additional polar factors. In wild-type cells, atleast a portion of ParA1, ParC, and FlhG is found withinpolar foci at either one or both poles, depending on theage of the cell. However, in a strain lacking
hubP
, thedistribution of these three proteins was markedly differ-entthaninwild-typecells(Fig.1B–E).ParA1wasdiffuselydistributed throughout the cell (Fig. 1B), and FlhG waseither diffusely distributed or found in a nonpolar focus(Fig. 1C); neither protein formed polar foci. ParC formedpolar foci in a subset of
hubP
cells (Fig. 1D); however,
;
50% of cells contained nonpolar ParC foci either in-stead of or in addition to a polar focus. An increase indiffusely distributed ParC was also evident in the
hubP
cells. Thus, HubP appears to be important for the propersubcellular distribution of three ParA-type ATPases in
V. cholerae
. However, it is not required for the correctcellular targeting of all such paralogous proteins. The
A multidomain hub organizes the cell poleGENES
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distribution of ParA2, which mediates chrII partitioning,and of MinD, which likely specifies the
V. cholerae
division site, was not altered in the absence of HubP(Supplemental Fig. S1). Additionally, the absence of HubPdid not cause detectable changes in cell shape or cell sizeand did not impair cell growth.Sequence and genomic analyses revealed several nota-ble attributes of the HubP protein. HubP is quite large(1621 amino acids, or
;
178 kDa) and extremely acidic(pI
=
3.22). Its N terminus appears to contain a signalsequence, a potential LysM peptidoglycan (PG)-bindingdomain, and a single transmembrane domain. Its Cterminus, which is predicted to reside within the cyto-plasm, contains 10 copies of an imperfect 46-amino-acidrepeat, in which 19 of the consensus amino acids areacidic(Fig.2A;SupplementalFig.S2).Theproteinisfairlywell conserved among vibrio and photobacteria species(
Vibrionaceae/Photobacteriaceae
); for example, homo-logs with
>
40% identity are present in
Vibrio vulnificus
and
Vibrio parahaemolyticus
, and
Vibrio fischeri
encodesa protein with 27.5% identity. Homologs appear to havesimilar overall structures, although the sizes and copynumbers of their acidic repeat sequences vary signifi-cantly (Supplemental Table S1; Supplemental Fig. S2).Outside of the
Vibrionaceae
, homologs are found princi-pally in the
g
-proteobacteria, where they are significantlyless conserved, but many appear to be encoded withina similar chromosomal neighborhood. Some of theseproteins (e.g., FimV of
Pseudomonas aeruginosa
) havebeen found to be associated with the assembly of type IVpili and pilus-associated twitching motility (Semmleret al. 2000; Wehbi et al. 2011).
Figure 1.
HubP is a determinant of polar proteinlocalization. (
A–D
) Subcellular localization of po-lar proteins fused to YFP (pseudocolored in greenwith phase contrast in the
top
panels) in indicated
V. cholerae
strains. (
A
) ParA1[K11E]. (
B
) ParA1. (
C
)FlhG. (
D
) ParC. Pictures shown are representativefields. Bar, 2
m
m. (
E
) Percentage of cells withmislocalized polar proteins. For ParA1[K11E],ParA1, and FlhG, mislocalization refers to cellslacking a polar focus, and for ParC, it refers to cellsthat contain a nonpolar focus. Means, standarddeviations, and total number of cells counted (
n
)are shown. See also Supplemental Fig. S1.
Yamaichi et al.2350 GENES
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HubP is directed to the
V. cholerae
pole and mid-cell by its periplasmic domain
Given the effect of HubP deficiency on subcellulartargeting of polar proteins, we assessed whether HubPitself is present at the
V. cholerae
cell poles. For theseexperiments, we used fluorescence microscopy and visu-alized either ectopically or chromosomally expressedHubP-CFP (or YFP/GFP). The fusion proteins, which arefunctional,astheydirectParA1[K11E]-YFPtoapolarposi-tion in the
hubP
mutant, displayed a bipolar distribution,even in small (young) cells (Fig. 2B–D). Thus, HubPappears to mark the new pole prior to the arrival of theParA family proteins, which are typically not bipolar inyoung cells (Fogel and Waldor 2006; Ringgaard et al.2011).HubPwasalsodetectableatthemid-cellinasubsetofcells,whereitalwayscolocalizedwithFtsZ,suggestingthat it arrives at this site as cells prepare to divide (Fig.2E). Time-lapse analyses confirmed that HubP is notpresent at the mid-cell in young cells, but instead arrivesthere as cells progress through the cell cycle, whichpresumably enables it to be present at newly formed
Figure 2.
HubP localization and its determinants. (
A
) Schematic of the HubP polypeptide (
top
) and truncation mutants (
bottom
) usedin this study. (Scissors) Signal sequence; (LysM) LysM domain; (TM) transmembrane domain; (10
3
repeat) repeat sequence (see thetext). (
B
) Subcellular localization of HubP-CFP in
D
hubP V. cholerae
. (
C
,
top
) Time-lapse images of plasmid-borne HubP-YFP in
D
hubPV. cholerae
. A schematic representation is drawn
below
. (
D
,
E
) Subcellular localization of HubP-CFP and ParA1[K11E]-YFP (
D
) and FtsZ-YFP (
E
) in
D
hubP V. cholerae
. In
E
, HubP and FtsZ at mid-cell are indicated with arrowheads and arrows, respectively. (
F
) FRAPexperiment of HubP-GFP. Pictures from a representative experiment (
left
) are shown, along with a graph of average signal intensitiesover time, based on analysis of 30 cells (
right
). The bleached focus is indicated by an arrow in the pictures and by the green line in thegraph. The magenta line shows the average intensity of the focus at the pole opposite the bleached pole. Pale blue and yellow lines showthe intensities of polar foci from unbleached (control) cells. (
G
) Fluorescence of -CFP and -mCherry fusions to truncated HubP in
V.cholerae
. Representative fields are shown. Bar, 2
m
m.
A multidomain hub organizes the cell poleGENES
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DEVELOPMENT 2351
poles (Fig. 2C). HubP localization was similar usingplasmid- and chromosome-encoded protein, although thechromosome-encoded protein was less abundant (Supple-mental Movies S1, S2).Fluorescence microscopy was also used to explore thedynamics and determinants of HubP’s subcellular local-ization. Notably, photobleaching of polar HubP-GFP wasfollowed by recovery of the polar signal, revealing thatHubP can be directly targeted to the pole; it does notneed to arrive there via the cell division site. Recoveryof fluorescence intensity at the bleached pole was accom-paniedbyadeclineoffluorescenceintensityattheopposite(unbleached) pole, suggesting that there is an exchange of HubP between the two poles (Fig. 2F). Additionally, wefound that polar targeting of HubP is independent of itsputative cytoplasmic and membrane-spanning domains.A fusion protein consisting of the first 324 amino acids of HubP (the putative periplasmic and transmembrane se-quences) fused to CFP had a cellular distribution equal tothat of the full-length fusion protein (Fig. 2G). A smaller(1–284)fragmentfusedtoCFPwasnotdetected,consistentwith the predicted localization of the HubP N-terminaldomain in the periplasm (Fig. 2A), where CFP does notfluoresce. However, when smaller N-terminal fragmentswere fused to mCherry (which is fluorescent even in theperiplasm), polar targeting was also observed (Fig. 2G,1–284). The first 161 amino acids of HubP were sufficientto enable polar targeting of the fusion protein, althoughthe first 150 amino acids were not (Fig. 2G, 1–161 and1–150). We also found that polar targeting of HubP re-quired the putative LysM domain (amino acids 90–134)(Fig. 2G,
D
LysM). In the absence of targeting sequences,HubP appeared to be diffusely distributed in the periplasm(Fig.2G,1–150and
D
LysM).TherequirementfortheLysMdomain raises the possibility that an interaction betweenHubP and PG contributes to its targeting and/or retentionat the
V. cholerae
pole. However, to date, we have beenunabletodetectadirectinteractionbetweenHubPandPGusing in vitro binding assays.
HubP modulates the localization of oriCI
We explored whether the failure of ParA1 to localize tothe cell poles in cells lacking HubP altered chrI segrega-tion dynamics. Although ParA1 is not essential for chrIpartitioning, in its absence,
oriCI
and the srcin-associ-ated centromere-binding protein ParB1 do not localize tothe old pole in young cells, nor are they drawn to the newpolefollowingchromosomereplication(FogelandWaldor2006). Instead, in a
parA1
deletion mutant, they aregenerally found at the mid-cell of young cells and nearthe 1/4 and 3/4 positions following replication (Fogel andWaldor 2006). We found that deletion of
hubP
has asimilar effect on the localization of ParB1: No polarParB1-CFP foci were detected in the
hubP
mutant, andcells typically contained two well-separated cytoplasmicfoci (Fig. 3A–C), as observed in
parA1
mutants. Thus,marking of the
V. cholerae
cell pole by HubP is critical forthe establishment of the normal cellular distribution of the organism’s major chromosome.
HubP binds directly to ParA1 to control its subcellular distribution
Several lines of evidence suggest that HubP controlsParA1’s distribution via a direct interaction between thetwo proteins. First, when ParA1[K11E]-YFP and HubP-CFPwerecoexpressedin
V.cholerae,
thefocitheyformedcolocalized (Fig. 2D; see below). Second, these proteinswere found to interact in a bacterial two-hybrid assay,which also showed that ParA1 can self-associate (Table 1;
Figure 3.
HubP governs the subcellular distribution of ParA1.(
A–C
) Subcellular localization of ParA1-YFP and ParB1-CFP inwild type (
A
) and
D
hubP
(
B
)
V. cholerae
cells. (
C
) The percentageof wild-type and
D
hubP
cells lacking polar foci of ParA1 (green)and ParB1 (magenta) is shown, based on analysis of the number of cells (
n
) indicated
above
. Asterisk indicates 0%. (
D
) Fluorescenceof HubP-CFP expressed in
E. coli
. (
E
,
F
) ParA1[K11E]-YFP (
E
) orParA1-YFP (
F
) was expressed in
E. coli
in the absence (
left
) orpresence (
middle
) of HubP-CFP (
right
). ParA1-YFP localized tothe nucleoid in the cell lacking HubP-CFP expression (markedwith an asterisk). Representative fields are shown. Bar, 2
m
m.
Yamaichi et al.2352 GENES
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